LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Experimenting With the Connections

When the heat sink is unplugged from the circuit board, the LCD screen is turned off. When we unplugged the white wire that connected the circuit board to the heating block the temperature reading on the LCD screen dropped drastically.

Test Run

We first tested open PCR on October 18, 2012. We learned how to take accurate temperatures using the open PCR machine. Using open PCR we were able to make a polymerase chain reaction. In order for this to occur, open PCR had to send the DNA through different sets of temperatures to heat it up to separate the strands and expose the bases, then cool it down for the primers to bind to the sequences, and also heat it back up to attain an extension of the copy of the new DNA. Which was conducted in an hour and thirty minutes.

Protocols

Polymerase Chain Reaction
The open PCR machine makes many copies of a DNA segment. It allows for a large enough sample to be made in order to analyze the DNA. In this lab we made copies of a specific segment of DNA that would allow us to determine whether or not our patients had the gene for cancer.

Sample Procedures

Reagent

Volume

Template DNA (20ng)

0.2 μL

10 μM forward primer

1.0 μL

10 μM reverse primer

1.0 μL

GoTaq master mix

50.0 μL

dH2O

47.8 μL

Total Volume

100.0 μL

Here is the patient information:

92136 M 54 Years

62276 F 61 Years

Fluorimeter Measurements

How does the fluorimetry technique work?

The fluorimeter is an instrument that detects fluorescence; the quantity is related to the amount of fluorescent material and is indirectly proportional to the molecule being detected. The slide that enters the fluorimeter is coated with a rough layer of Teflon which allows drops of liquid to form beads on the surface due to the surface tension. Because of this, the light from the Blue LED is focused in the drop which increases the intensity, and the SYBR Green in the liquid causes the presence of DNA to be indicated through a green phosphorescence. In this procedure, two drops of SYBR Green were placed on the slide, and then two drops of the solutions from the samples being tested were placed on top of the SYBR Green. The fluorimeter will give a visual color signal when dsDNA is present, and this can be quantified by taking an image. This is done by placing the smartphone camera (in this case, a Samsung HTC 1) into a diffraction grating and mounting it, and then setting exposure and saturation levels. By the use of a self-timer, the least amount of outside light was allowed in.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

A Polymerase chain reaction is a machine that amplifies a single or a few strands of DNA to generate millions of copies of that DNA sequence. Using this technology scientists can determine whether a patient has a positive or negative result towards cancer. A method of getting this data is called the PCR detection method, a method that relies on thermal cycling, switching back and forth to melt DNA and then connect primers. This is a method that can be used to detect whether a patient has positive result for cancer, because a sample of DNA can be taken and whether that connects to the primers and creates a chain reaction, scientists can then determine whether this DNA is positive or negative towards cancer. An example of proving this method can be seen using the r17879961 SNP, a cancer-associated sequence, using the PCR detection method we can prove that r17879961 SNP is actually associated with cancer. Because it carries with the Polymerase chain reaction, and to further prove the patient has a positive result for cancer, we use fluorescent dye and if the DNA glows in the solution, then the results are positive for cancer.

(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)